CN111012783A - Application of autophagy inhibitor in preparation of drugs for inhibiting basophil activation - Google Patents
Application of autophagy inhibitor in preparation of drugs for inhibiting basophil activation Download PDFInfo
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- CN111012783A CN111012783A CN201911146490.5A CN201911146490A CN111012783A CN 111012783 A CN111012783 A CN 111012783A CN 201911146490 A CN201911146490 A CN 201911146490A CN 111012783 A CN111012783 A CN 111012783A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P37/08—Antiallergic agents
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Abstract
The invention discloses application of an autophagy inhibitor in preparation of a medicament for inhibiting basophil activation. At present, an ideal method or medicament for inhibiting the activation of basophils is not available in the market, and the inventor of the application finds that the inhibition of autophagy can inhibit the anti-IgE mediated activation of human basophils, and is expected to provide a new strategy for clinically inhibiting the activation of basophils and preventing and treating diseases such as allergy, autoimmunity and the like.
Description
Technical Field
The invention relates to the technical field of immune regulation, in particular to application of an autophagy inhibitor in preparation of a medicament for inhibiting basophil activation.
Background
Autophagy (Autophagy) is a highly conserved metabolic pathway in the life process of eukaryotes, is a lysosome-mediated process for degrading intracellular misfolded proteins and aged or damaged organelles and the like, and is important for survival, differentiation, development and homeostasis of cells. Autophagy plays an important regulatory role in innate and adaptive immune processes, including recognition and killing of pathogens, antigen presentation, lymphocyte differentiation and development, immunoregulation, and the like.
Basophils (Basophils) are a subset of peripheral blood leukocytes, and in recent years, the role of Basophils in innate and adaptive immunity, particularly in immunomodulation, has attracted considerable attention from researchers, and research on such cells has led to significant progress and attention. Activation is a key step in the development of biological functions by basophils, and various pathways mediate the activation of basophils and produce various types of effector molecules, which are then further developed through various pathways. In both allergic and autoimmune diseases, it has been found that activated basophils promote disease progression.
At present, the strategy for inhibiting basophil activation mainly comprises (1) anti-IgE antibody, which is based on the principle that free IgE is bound, so that basophil activation mediated after IgE is bound with a high affinity receptor Fc epsilon RI is reduced, and then rapid degranulation caused by basophil activation is reduced, active substances such as histamine and leukotriene C4 are released, and the anti-IgE antibody can be used for treating allergic diseases such as bronchial asthma and chronic urticaria, wherein the scheme has the main defects that ① has no specificity, is bound with all IgE, but not the specific IgE of an allergen, and can induce the need of inducing the allergic diseases such as bronchial asthma and chronic urticariaFor diseases requiring IgE antibody to participate in protection, such as parasitic infection, etc., it is reported that non-autoreactive IgE, unlike autoreactive IgE, has pathogenicity, can produce IFN- α by inhibiting TLR 7/9-mediated plasmacytoid dendritic cells, and can play a negative regulation role in SLE pathogenesis, ② can only eliminate free IgE, has no effect on IgE bound with cells, and is not suitable for treating diseases after basophils are activated, ③ has obvious side effect, and has various clinical adverse effects, especially arterial thrombosis events, cancer risks and the like (caused by the fact that the IgE is not bound with cells
Instructions provide or FDA warning information: xolair Side Effects May Cause HeartProblmes, Cancer: FDA waves). (2) Is a chemical small molecule inhibitor of basophil activation which is still under development. Mainly targets a series of key kinases of downstream signal molecules of an IgE-mediated basophil activation signal channel. At present, all of the chemical small molecule inhibitors are still in the development stage and cannot be used in clinic. Disadvantages include that the specificity of these small molecule compounds is still to be improved, that part of the target sites are too high, and that the strategy for inhibiting a single kinase site is still to be optimized, etc.
In view of the above, there is currently no ideal method for inhibiting basophil activation.
Disclosure of Invention
In view of the above problems, the present invention aims to overcome the disadvantages of the prior art and to provide a method for effectively inhibiting the activation of basophils.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following aspects:
in a first aspect, the invention provides the use of an autophagy inhibitor for the manufacture of a medicament for inhibiting basophil activation.
Preferably, the autophagy inhibiting agent is chloroquine or Atg7 siRNA.
More preferably, the basophils are activated by anti-IgE stimulation.
In another aspect, the invention provides the use of an autophagy inhibitor for the preparation of a medicament for the prevention or treatment of an allergic and/or autoimmune disease.
Preferably, the autophagy inhibiting agent is chloroquine or Atg7 siRNA.
In yet another aspect, the present invention provides an agent for inhibiting basophil activation, comprising chloroquine or/and Atg7 siRNA.
In conclusion, the beneficial effects of the invention are as follows:
at present, an ideal method or medicament for inhibiting the activation of basophils is not available in the market, and the inventor of the application finds that the inhibition of autophagy can inhibit the anti-IgE mediated activation of human basophils, and is expected to provide a new strategy for clinically inhibiting the activation of basophils and preventing and treating diseases such as allergy, autoimmunity and the like.
Detailed Description
The inventor of the application unexpectedly finds that the anti-IgE mediated activation of human basophils can be inhibited by inhibiting autophagy, and a new strategy is hopefully provided for clinically inhibiting the basophil activation, preventing and treating allergy, autoimmune diseases and the like.
In some embodiments, the present invention provides a method of inhibiting anti-IgE-mediated basophil activation, comprising the steps of:
preparing basophilic granulocyte from anticoagulated peripheral blood of a normal person by using a negative selection method, transfecting by using an autophagy inhibition drug chloroquine or Atg7siRNA, and inhibiting autophagy of the basophilic granulocyte;
then stimulating and activating basophilic granulocyte by anti-IgE, and detecting the expression of the activation indexes of CD203c, intracellular IL-4, IL-6 and IL-13. As a result, after the inhibition of autophagy, the activation of basophil mediated by anti-IgE is obviously inhibited.
In some preferred embodiments, the present invention provides a method of inhibiting anti-IgE mediated basophil activation, comprising the steps of:
(1) basophilic granulocytes were prepared from anticoagulated peripheral blood of normal humans by the negative selection method:
firstly, collecting EDTA or heparin anticoagulated peripheral venous blood after obtaining the informed consent of volunteers; secondly, high-purity basophils are prepared by using a commercial human basophil negative selection kit and diluted to (1.0-8.0) multiplied by 10 by using a cell culture medium5Per ml;
(2) transfection of the drug chloroquine for autophagy inhibition, or Atg7siRNA, for inhibition of human basophil autophagy:
treating the basophils prepared above with chloroquine (0.1-20 μmol/L) as autophagy inhibiting drug for 12 hours, or transfecting Atg7siRNA into the basophils with a commercial siRNA transfection kit, and then continuing culturing for 20 hours;
(3) then stimulating and activating basophilic granulocytes by anti-IgE, and detecting that the activation indexes are CD203c and the expressions of intracellular IL-4, IL-6 and IL-13:
adding anti-IgE (0.1-10 μ g/mL) into the culture solution of basophil for stimulating for 24 hr, and adding agent for inhibiting cytokine secretion (BFA/Monensin mixture) 12 hr before the culture; using PBS (containing 135mM NaCl,4.7mM KCl,10mM Na)2HPO4，2mM NaH2PO4pH 7.4), adding a fluorescein-labeled antibody combination (anti-CD123, anti-CD203c, anti-IL-4, anti-IL-6 and anti-IL-13) for identifying basophils, incubating for 30 minutes, washing by PBS, and then loading to a flow cytometer to detect and analyze the influence of autophagy intervention on anit-IgE-mediated activation of human basophils;
(4) after inhibition of autophagy, Anti-IgE mediated activation of basophils was found to be inhibited: analysis of the detection results shows that after transfection of the medicament chloroquine for inhibiting autophagy or Atg7siRNA for inhibiting human basophil autophagy, the expression level of basophil activation markers (CD203c, IL-4, IL-6 and IL-13) can be remarkably reduced.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. Unless otherwise specified, the reagents and materials of the present invention are commercially available or may be obtained from other publicly available sources.
Example 1
(1) First, after obtaining informed consent from healthy volunteers, 20ml of EDTA-anticoagulated peripheral venous blood was collected and subjected to negative selection with a commercial human basophil kit (EasySep)TMHuman Basophil Isolation Kit, Stemcell, USA) to prepare Basophil with purity of 95%, and diluting to 5.0 × 105One per ml.
(2) Basophils prepared above were treated with chloroquine (1.0. mu. mol/L, Sigma, USA), an autophagy-inhibiting drug, for 12 hours.
(3) In the above basophil culture solution for inhibiting autophagy, anti-IgE (1.0 μ g/mL) was added continuously for 24 hours, and a reagent for inhibiting cytokine secretion (BFA/Monensin mix) was added 12 hours before the end of the culture; using PBS (containing 135mM NaCl,4.7mM KCl,10mM Na)2HPO4,2mM NaH2PO4pH 7.4), adding a fluorescein-labeled antibody combination (anti-CD123, anti-CD203c, anti-IL-4, anti-IL-6, anti-IL-13) recognizing basophils, incubating for 30 minutes, washing with PBS, and then loading into a flow cytometer to detect and analyze the effect of autophagy intervention on anit-IgE-mediated activation of human basophils.
(4) Analysis of the detection results shows that after the inhibition of human basophil autophagy by using the medicament chloroquine for inhibiting autophagy, the expression levels of basophil activation markers (CD203c, IL-4, IL-6 and IL-13) can be remarkably reduced (the results are shown in the following table 1).
TABLE 1 Effect of chloroquine inhibition of autophagy on anti-IgE mediated basophil activation
Example 2
(1) First, after obtaining informed consent from healthy volunteers, 30ml of heparin-anticoagulated peripheral venous blood was collected, and purified by a commercial human Basophil negative selection Kit (human,miltenyi biotec, USA) was prepared in 97% purity, and diluted to 8.0 × 10 concentration5One per ml.
(2) Using a commercial siRNA transfection kit (
RNAimax Reagent, ThermoFisher USA) to transfect Atg7siRNA (Santa Cruz, USA) to basophils, and then culture was continued for 20 hours.
(3) Continuously adding anti-IgE (5.0 mu g/mL) into the culture solution of basophilic granulocyte for 24 hours, and adding a reagent for inhibiting cytokine secretion (BFA/Monensin mixture) 12 hours before the culture is finished; using PBS (containing 135mM NaCl,4.7mM KCl,10mM Na)2HPO4,2mM NaH2PO4pH 7.4), adding a fluorescein-labeled antibody combination (anti-CD123, anti-CD203c, anti-IL-4, anti-IL-6, anti-IL-13) recognizing basophils, incubating for 30 minutes, washing with PBS, and then loading into a flow cytometer to detect and analyze the effect of autophagy intervention on anit-IgE-mediated activation of human basophils.
(4) Analysis of the test results shows that the expression levels of basophil activation markers (CD203c, IL-4, IL-6 and IL-13) can be remarkably reduced after transfection of Atg7siRNA to inhibit the autophagy of human basophils (see the results in the following table 2).
TABLE 2 Effect of Atg7siRNA transfection on inhibition of autophagy on anti-IgE-mediated basophil activation
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. Use of an autophagy inhibitor for the preparation of a medicament for inhibiting basophil activation.
2. The use of claim 1, wherein the autophagy inhibiting agent is chloroquine or Atg7 siRNA.
3. Use according to claim 1, wherein the basophils are activated by anti-IgE stimulation.
4. Use of an autophagy inhibitor for the preparation of a medicament for the prevention or treatment of an allergic disease and/or an autoimmune disease.
5. The use of claim 4, wherein the autophagy inhibiting agent is chloroquine or Atg7 siRNA.
6. An agent for inhibiting basophil activation, comprising chloroquine or/and Atg7 siRNA.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911146490.5A CN111012783A (en) | 2019-11-20 | 2019-11-20 | Application of autophagy inhibitor in preparation of drugs for inhibiting basophil activation |
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| CN201911146490.5A CN111012783A (en) | 2019-11-20 | 2019-11-20 | Application of autophagy inhibitor in preparation of drugs for inhibiting basophil activation |
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| CN201911146490.5A Pending CN111012783A (en) | 2019-11-20 | 2019-11-20 | Application of autophagy inhibitor in preparation of drugs for inhibiting basophil activation |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1470237A (en) * | 2002-06-28 | 2004-01-28 | Use of chloroquine, hydroxychloroquine and 4-aminoquinazine derivatives in preparing medicine for antiretrovirus therapy |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1470237A (en) * | 2002-06-28 | 2004-01-28 | Use of chloroquine, hydroxychloroquine and 4-aminoquinazine derivatives in preparing medicine for antiretrovirus therapy |
Non-Patent Citations (3)
| Title |
|---|
| BETTINA WEDI等: "Chronic urticaria serum induces histamine release, leukotriene production, and basophil CD63 surface expression—Inhibitory effects of anti-inflammatory drugs", 《J ALLERGY CLIN IMMUNOL》 * |
| ELHAM TAHERIAN等: "The Biological and Clinical Activity of Anti-Malarial Drugs In Autoimmune Disorders", 《CURRENT RHEUMATOLOGY REVIEWS》 * |
| 陈钟英等主编: "《临床药物手册（第三版）》", 31 August 1995, 上海科学技术出版社 * |
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